Insect cells have been successfully used to replicate baculoviruses to promote expression of foreign genes carried by baculoviruses. [Smith et al., PNAS (U.S.A.), 82:8404-8408 (December, 1985) wherein Spodoptera frugiperda cells infected with recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) carrying cDNA coding for human IL-2 are reported to produce high levels of IL-2; see also, Smith et al., European Patent Application Publication No. 127,839 (published Dec. 12, 1984), wherein methods for producing recombinant baculoviruses capable of expressing selected genes in host insect cells are disclosed; and Jeang et al., J. Virol., 61 (3):708-713 (March 1987), wherein the production of functional human T-cell leukemia virus type I (HTLV-I) p40.sup.x protein by S. frugiperda (Sf9) cells infected with a recombinant AcNPV virus is reported.]
Insect cells have been cultured for the production of insect viruses as biological insecticides. [Vaughn et al., In Vitro, 13:213-217 (1977); Lynn et al., J. Invert. Pathol., 32:1-5 (1978)]. Such viruses include, for example, baculoviruses and non-baculoviruses such as infectious flacheriae virus (IFV) and cytoplasmic polyhedrosis virus (CPV). Exemplary are certain baculoviruses, for example, nucleopolyhedrosis viruses (NPV) and granulosis viruses (GV), which are highly virulent for pest insects; some of the most promising have been commercially developed as biological pesticides pathogenic for agriculturally important insects. [Burges (ed.), Microbial Control of Pests and Plant Diseases 1970-1980 (London, 1981); Miltenburger et al., Bioinsecticides II: Baculoviridae. Adv. Biotechnol. Processes, 3:291 (1984); for a discussion of such NPV and GV products as biological pesticides, see Shieh et al., "Production and Efficiency of Baculoviruses," Biotechnology and Bioengineering Vol. XXII, 1357 (1980); see also Huber, "Use of Baculoviruses in Pest Management Programs," In Granados et al., (eds.), The Biology of Baculoviruses: Vol. II Practical Applications for Insect Control, pp. 181-202 (1986).]
Baculoviruses are very stable and are able to persist for longer times in the environment than other animal viruses. This unusual biological stability is the result of a unique association of the infectious virus particles and a viral occlusion which is a crystalline assembly of a viral encoded structural protein called polyhedrin. Late in viral replication, baculovirus particles become embedded in a protein occlusion composed of the polyhedrin protein. Insects acquire a baculovirus disease by ingesting these occluded virus (OV) contaminating their food supply. The polyhedrin matrix protects the virus particles outside of the insect and also during their passage through the foregut of the insect. In the insect midgut, the alkaline pH activates the dissolution of the polyhedrin crystalline matrix resulting in the release of many viruses. The virus becomes absorbed by the midgut epithelial cells initiating the infection process. There is a second infectious form of nuclear polyhedrosis viruses (NPVs), known as the extracellular or nonoccluded virus (NOV) form, which is generated by the budding of viral nucleocapsids through the plasma membrane of the infected cells. NOVs are responsible for spreading the secondary infection via the hemolymph of the insect.
Traditionally, production of baculoviruses was achieved using insect larvae; however, large sale production by such means is not attractive. Insect cell culture is much more practical. [Vaughn, Adv. Cell. Cult., 1:281-295 (1981); Stockton et al., In Burges (ed.), supra, at 313-328.] Batchwise and semicontinuous production of Spodoptera frugiperda and Trichoplusia ni cells that allow the replication of Autographa californica nuclear polyhedrosis virus have been reported. [Vaughn, J. Invert. Path., 28:233-237 (1976); Hink, In Kurstak (ed.), Microbial Viral Pesticides at 493-506 (1982).]
To obtain the maximum yield of insect cells in culture with minimum population doubling time, the cells must be provided with ideal nutritional, biological, and biophysical requirements for growth. One of the most important variables is the composition of the insect cell culture media. The basis of a commonly employed medium f 15 for lepidopteran cells was a medium developed by Wyatt, J. Gen. Physiol., 39:841 (1956) and modified by Grace, Nature (London), 195:788 (1962). That medium
resembles hemolymph and consists mainly of chemically pure amino acids, vitamins, organic acids and inorganic salts, and originally was supplemented with insect hemolymph. Later the hemolymph from this formulation was replaced by fetal bovine serum, bovine serum albumin, and whole-egg ultrafiltrate. PA0 [Weiss et al., Chapter 3, page 68, In Granados et al. (eds), The Biology of Baculoviruses (Vol. II) 1986; citations omitted.] PA0 Research findings on the role and utilization of amino acids, minerals, hormones, mitogens, carbohydrates, organic acids, fatty acids, sugars, lipids, and monovalent and divalent cations in cultured insect cells have led to some experimental application of serum-free media for insect cells. However, to date these serum-free media are not completely satisfactory for viral replication, which may be attributed to the absence of undefined proteins normally in serum supplements. PA0 [Citations omitted.] PA0 The mechanical strength of insect cells in culture is small . . . This has definite consequences for the scale up of insect cell cultures. Larger volumes of insect cell cultures require more efficient oxygen transfer to the solution than can be achieved by flushing air/oxygen over the liquid surface. However, dispersion of gas by means of stirring and sparging air through the cell suspension to provide sufficient oxygen probably results in a larger decay rate than growth rate of the cells.
The cultivation of insect cells in vitro at large scale, infecting the cells with either recombinant or non-recombinant viruses, and work-up of the recombinant product or the virus material depends on the availability of a suitable culture medium. As indicated above, conventional culture media contain, in addition to inorganic salts, vitamins, amino acids, sugars, and a number of other organic compounds important for cell physiology, about 2-25% by weight per volume of animal serum and/or animal serum albumin. Animal serum is a limiting factor due to its high price and relatively scarce availability, and further, because of the complications it creates for product purification. Production of insect cells on a scale required for either the industrial manufacture of insect pathogenic viruses or of recombinant products necessitates the replacement of animal serum by a cheaper component available in sufficient quantities.
There have been a number of studies done to develop serum-free media for the cultivation of insect cells, many of which are in the context of producing NPV and GV as biological pesticides and which include the following: Goodwin et al., in Kurstak et al. (eds.) Invertebrate Systems In Vitro, Chapter 45:493-509 (1980); Goodwin, In Vitro, 12:303-304 (1976); Hink et al., In Vitro, 13:177 (1977); Roder, U.S. Pat. No. 4,454,227; Weiss et al., In Vitro, 20:271 (1984); Gardiner et al., J. Invert. Path., 25:363-370 (1970); Wilkie et al., Develop. Biol. Standard., 46:29-37 (1980); and Vail et al., J. Invert. Path., 28:263-267 (1976).
Table 1 lists a number of the media wherein serum was replaced by protein hydrolyzates and crude and defined lipids. Such serum-free media were reported to support the production of baculovirus in insect (Lepidopteran) cells although some of the media did not produce viable occluded virus.
TABLE 1 __________________________________________________________________________ Lepidopteran Serum-Free Culture Report Cell Line Medium __________________________________________________________________________ Hink et al. In Vitro, Trichoplusia ni Defined medium 13:177 (1977) + Bactotryptose Goodwin et al., In Vitro, Porthetria dispar Defined medium 12:303 (1976) + liver digest, peptic peptone, yeast extract, Lactalbumin Hydrolysate Roder, Naturwissenschaften, Spodoptera frugiperda Defined medium + 69:92 (1982) peptones, egg yolk Weiss et al., In Vitro, Heliothis zea RIL-2B medium + 20:271 (1984) sterols, fatty acids Goodwin et al., Invert. Lymantria dispar Defined medium + Systems, Chap 45:493 peptones, glutamine, (1980) glycerol, .alpha.-glycero- phosphate, oleate, Tween 80, cholesterol, .alpha.- Tocopherol __________________________________________________________________________
In regard to such serum free media, Weiss et al. points out in The Biology of Baculoviruses, (CRC Press 1986) supra, at page 68 that:
In the context of culturing insect cells for the production of recombinant proteins via a baculovirus expression vector system (BEVS), Miller et al. [In: Setlow et al. (eds.), Genetic Engineering Principles and Methods (Vol 8): "An Insect Baculovirus Host-Vector System for High-Level Expression of Foreign Genes," pp. 277-298 (1986)] states at page 291: "Typically 10% fetal calf serum is present throughout cell and viral growth. The protein contributed by the serum can be a problem for purification and perhaps expression if it contains certain antagonistic activities. While the literature contains reports of insect cells growing in serum-free media, such media do not readily support viral growth." Miller et al. then notes that U.S. Pat. No. 4,454,227 to Roder wherein an egg yolk emulsion is used as a serum replacement is an "interesting exception" but that it "is not clear that it eases purification problems. As a short term solution, we have observed that, once infected in 10% serum-containing media, cells can be transferred to a serum-free media and expression will occur largely unabated." However, such a transfer from serum-containing medium to a serum free medium, as suggested by Miller et al., is not a practical solution for large scale industrial production.
Summers et al. in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures" Texas Agricultural Experiment Bulletin No. 1555 (Texas A & M University; May 1987) recommends, at pages 32-33, the use of "Grace's Antheraea medium (a relatively simple mixture of salts, carbohydrates and amino acids) . . . used for short term incubations of cells (rinsing monolayers, seeding cells, transfection, etc.)" and "TNM-FH, a more complete medium suitable for routine growth of cells in monolayer or suspension, . . . prepared from Grace's by the addition of 3.3 g/liter Yeastolate and 3.3 g/liter Lactalbumin Hydrolysate (both available from Difco)." Summers et al. goes on to state at page 33 that for "complete growth medium, add 10% fetal bovine serum (sterile, heat inactivated", and under the Methods section at pages 9 and 11, Summers et al. directs that "TNM-FH media+10% FBS+antibiotics" be used. At page 10, Summers et al. states: "It is desirable to seed cells in serum-free medium to promote rapid and firm attachment for plaque assays, infections, etc. However, cells may begin to show signs of stress if they are left without serum for more than 2 hours."
Most culture media for the growth of insect cells contain peptones and serum and are conventionally used to grow insect cells under poorly aerated conditions which restrict the cells' growth and expression of recombinant proteins and viable viral particles. Although serum-free media have been developed that support insect cell growth, none of such media have been shown to support production of levels of recombinant proteins from insect cells grown in such serum-free media and infected with a recombinant baculovirus, comparable to levels achieved from insect cells so infected and grown in approximately 10% serum-containing media. Further, as indicated above, none have been shown to support viral replication in insect cells satisfactorily.
Attempts to include lipid nutrients in insect culture media have often resulted in the presence of large insoluble lipid particles not easily available to cells. This invention solves that problem.
The research described herein indicates that some of the peptones and other ingredients included in published insect culture media, both serum and non-serum containing, are growth inhibitory or unnecessary. Further, the research indicates that high molecular weight components of the peptone fractions may interfere with the purification of the recombinant or viral products produced by the cultured insect cells; this invention provides a means to remove such components.
Further, of great value to the art would be a single serum-free, low or no protein medium for the growth of insect cells and production of recombinant proteins or viral products thereby respectively either via a baculovirus expression vector system (BEVS) or by infection with a wild-type virus. On a small scale, insect cells can be grown in a serum-containing relatively high protein medium, and can then be separated from such growth medium and resuspended in a serum-free production medium for infection and expression of products. Such separation and resuspension processes are not desirable for large scale culture because of the complexity of the equipment and operations required for such procedures. There is a need in the art for a single serum-free and low or no protein growth and production medium which would simplify fermentation operations, reduce the cost of large scale insect cell culture, and aid in the purification of the recombinant and viral products. This invention meets that need.
A reason that poorly aerated conditions have been conventionally used in insect cell culture is because of "the fragility of insect cells". [Weiss et al., The Biology of Baculoviruses, Vol. II (Chapter 3) at page 80 (CRC Press 1986); Weiss et al., In Vitro, 16:222 (1980).] "Two problems appear to have delayed the full utilization of suspension systems for the large volume culture of insect cells: the fragility of insect cells, and second, the high oxygen demand, particularly for virus-infected cells." [Weiss et al., CRC Press, supra, p. 80; citations omitted.] Tramper et al., Enzyme Microb. Technol., 8:33-36 (January 1986) notes at p. 33: "A major problem encountered in scaling up [insect] cell culture systems is the shear sensitivity of these cells due to their size (20 .mu.m range) and lack of cell wall. The shear sensitivity may hamper the supply of sufficient oxygen in a conventional manner (e.g., by sparging)." Tramper et al. further states at pages 35-36:
The instant invention meets the need of providing a medium in which insect cells can grow not only under the poorly aerated conditions of conventional culture, but in well-aerated conditions wherein insect cells can grow to high cell density with high viability and produce levels of recombinant products, when infected with recombinant baculoviruses, or of other viral products, when infected with wild-type viruses, comparable to levels achieved in serum-containing media.